CN112461943A - High performance liquid chromatography detection method for immunoglobulin igG in cow milk - Google Patents
High performance liquid chromatography detection method for immunoglobulin igG in cow milkDownload PDFInfo
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- CN112461943A CN112461943ACN202011085067.1ACN202011085067ACN112461943ACN 112461943 ACN112461943 ACN 112461943ACN 202011085067 ACN202011085067 ACN 202011085067ACN 112461943 ACN112461943 ACN 112461943A
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- 238000001514detection methodMethods0.000titleclaimsabstractdescription30
- 108060003951ImmunoglobulinProteins0.000titleclaimsabstractdescription22
- 102000018358immunoglobulinHuman genes0.000titleclaimsabstractdescription22
- 235000020247cow milkNutrition0.000titleclaimsabstractdescription19
- 238000004128high performance liquid chromatographyMethods0.000titleclaimsabstractdescription13
- 235000013336milkNutrition0.000claimsabstractdescription23
- 239000008267milkSubstances0.000claimsabstractdescription23
- 210000004080milkAnatomy0.000claimsabstractdescription23
- 238000000034methodMethods0.000claimsabstractdescription18
- QTBSBXVTEAMEQO-UHFFFAOYSA-NAcetic acidChemical compoundCC(O)=OQTBSBXVTEAMEQO-UHFFFAOYSA-N0.000claimsdescription30
- 101710120037Toxin CcdBProteins0.000claimsdescription20
- 239000012064sodium phosphate bufferSubstances0.000claimsdescription20
- 238000010828elutionMethods0.000claimsdescription16
- 239000000243solutionSubstances0.000claimsdescription15
- 239000006228supernatantSubstances0.000claimsdescription10
- 239000000047productSubstances0.000claimsdescription6
- 238000010790dilutionMethods0.000claimsdescription5
- 239000012895dilutionSubstances0.000claimsdescription5
- 238000001914filtrationMethods0.000claimsdescription5
- 239000012528membraneSubstances0.000claimsdescription5
- 238000000926separation methodMethods0.000claimsdescription5
- 238000005303weighingMethods0.000claimsdescription5
- 239000007788liquidSubstances0.000abstractdescription3
- 238000009417prefabricationMethods0.000abstract1
- 238000006467substitution reactionMethods0.000description2
- 239000007791liquid phaseSubstances0.000description1
- 230000000813microbial effectEffects0.000description1
- 239000012071phaseSubstances0.000description1
- 238000002798spectrophotometry methodMethods0.000description1
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/04—Preparation or injection of sample to be analysed
- G01N30/06—Preparation
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/26—Conditioning of the fluid carrier; Flow patterns
- G01N30/28—Control of physical parameters of the fluid carrier
- G01N30/34—Control of physical parameters of the fluid carrier of fluid composition, e.g. gradient
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/26—Conditioning of the fluid carrier; Flow patterns
- G01N30/28—Control of physical parameters of the fluid carrier
- G01N30/36—Control of physical parameters of the fluid carrier in high pressure liquid systems
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/60—Construction of the column
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- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/88—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86
- G01N2030/8809—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample
- G01N2030/8813—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample biological materials
- G01N2030/8831—Integrated analysis systems specially adapted therefor, not covered by a single one of the groups G01N30/04 - G01N30/86 analysis specially adapted for the sample biological materials involving peptides or proteins
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- Analytical Chemistry (AREA)
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Abstract
Description
Technical Field
The invention relates to a high performance liquid chromatography detection method for immunoglobulin igG in cow milk, belonging to the technical field of microbial detection.
Background
The existing standard detection method for detecting the immunoglobulin IgG in the milk is only a spectrophotometer method, and NY/T2070 and 2011 determination spectrophotometry for the immunoglobulin IgG in the milk and products thereof.
Therefore, in order to meet the current situation, a brand-new method for detecting the immunoglobulin in the cow milk needs to be developed to meet the actual use requirement.
Disclosure of Invention
The invention aims to provide a high performance liquid chromatography detection method for immunoglobulin igG in cow milk.
In order to achieve the purpose, the invention provides the following technical scheme:
a high performance liquid chromatography detection method for immunoglobulin igG in cow milk comprises the following steps:
s1, preparing a sample, namely accurately weighing milk to be detected, adding a sodium phosphate buffer solution into the milk for dilution, then performing centrifugal separation on the diluted milk through a centrifugal machine, standing, and finally taking a supernatant after standing and filtering the supernatant through an ionic membrane to obtain a finished product for later use;
s2, detecting the chromatographic column, namely selecting a chromatographic column-Bio Protein G column, adjusting the chromatographic column-Bio Protein G column to 10-45 ℃ at a constant speed, taking 8-15 mu L of the sample prepared in the step S1, adding the sample into the chromatographic column-Bio Protein G column at a constant speed of 0.3-0.6 mL/min, adding a sodium phosphate buffer solution (pH 7.4) with the concentration of 50mmol/L and acetic acid with the concentration of 0.5mol/L into the chromatographic column-Bio Protein G column at a constant speed, and performing gradient elution operation, namely performing detection operation in the gradient elution process.
Furthermore, in the step S1, the use ratio of the cow milk to the sodium phosphate buffer solution is 1: 2-10.
Further, in the step S2, only one of a sodium phosphate buffer (ph 7.4) and acetic acid is used in a single operation during the gradient elution.
The invention establishes a brand-new method for measuring the immune globulin IgG in the milk, effectively expands the types and types of the detection method of the immune globulin IgG in the milk, has high detection precision, provides more choices for the detection field, adopts a liquid chromatograph which has high popularization rate compared with the traditional detection, has good instrument operation stability and is easy to operate, and can effectively simplify the pretreatment step of detecting samples, thereby improving the detection efficiency.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the drawings without creative efforts.
FIG. 1 is a schematic structural view of the present invention;
FIG. 2 is a table of conditions for liquid phase mobile phase gradient elution.
Detailed Description
The technical solutions of the present invention will be described clearly and completely with reference to the accompanying drawings of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1
As shown in fig. 1, a method for detecting immunoglobulin igG in cow milk by high performance liquid chromatography comprises the following steps:
s1, preparing a sample, namely accurately weighing milk to be detected, adding a sodium phosphate buffer solution into the milk for dilution, then performing centrifugal separation on the diluted milk through a centrifugal machine, standing, and finally taking a supernatant after standing and filtering the supernatant through an ionic membrane to obtain a finished product for later use;
s2, detecting the chromatographic column, namely, firstly selecting a chromatographic column-Bio Protein G column, uniformly adjusting the chromatographic column-Bio Protein G column to 10 ℃, then taking 8 mu L of the sample prepared in the step S1, uniformly adding the sample into the chromatographic column-Bio Protein G column at a speed of 0.3mL/min, and then uniformly adding a sodium phosphate buffer solution (pH 7.4) with a concentration of 50mmol/L and acetic acid with a concentration of 0.5mol/L into the chromatographic column-Bio Protein G column to perform gradient elution operation, namely, performing detection operation in the gradient elution process.
In the step S1, the use ratio of the cow milk to the sodium phosphate buffer solution is 1: 2.
Meanwhile, in the S2, only any one of a sodium phosphate buffer (ph 7.4) and acetic acid was used in a single operation during the gradient elution.
Example 2
As shown in fig. 1, a method for detecting immunoglobulin igG in cow milk by high performance liquid chromatography comprises the following steps:
s1, preparing a sample, namely accurately weighing milk to be detected, adding a sodium phosphate buffer solution into the milk for dilution, then performing centrifugal separation on the diluted milk through a centrifugal machine, standing, and finally taking a supernatant after standing and filtering the supernatant through an ionic membrane to obtain a finished product for later use;
s2, detecting the chromatographic column, namely, firstly selecting a chromatographic column-Bio Protein G column, uniformly adjusting the chromatographic column-Bio Protein G column to 45 ℃, then taking 15 mu L of the sample prepared in the step S1, uniformly adding the sample into the chromatographic column-Bio Protein G column at a speed of 0.6mL/min, and then uniformly adding a sodium phosphate buffer solution (pH 7.4) with a concentration of 50mmol/L and acetic acid with a concentration of 0.5mol/L into the chromatographic column-Bio Protein G column to perform gradient elution operation, namely, performing detection operation in the gradient elution process.
In this embodiment, in the step S1, the ratio of cow milk to sodium phosphate buffer is 1: 10.
Meanwhile, in the S2, only any one of a sodium phosphate buffer (ph 7.4) and acetic acid was used in a single operation during the gradient elution.
Example 3
As shown in fig. 1, a method for detecting immunoglobulin igG in cow milk by high performance liquid chromatography comprises the following steps:
s1, preparing a sample, namely accurately weighing milk to be detected, adding a sodium phosphate buffer solution into the milk for dilution, then performing centrifugal separation on the diluted milk through a centrifugal machine, standing, and finally taking a supernatant after standing and filtering the supernatant through an ionic membrane to obtain a finished product for later use;
s2, detecting the chromatographic column, namely, firstly selecting a chromatographic column-Bio Protein G column, uniformly adjusting the chromatographic column-Bio Protein G column to 25 ℃, then taking 5 mu L of the sample prepared in the step S1, uniformly adding the sample into the chromatographic column-Bio Protein G column at a speed of 0.5mL/min, and then uniformly adding a sodium phosphate buffer solution (pH 7.4) with a concentration of 50mmol/L and acetic acid with a concentration of 0.5mol/L into the chromatographic column-Bio Protein G column to perform gradient elution operation, namely, performing detection operation in the gradient elution process.
In this embodiment, in the step S1, the ratio of cow milk to sodium phosphate buffer is 1: 5.
In this example, in S2, only one of sodium phosphate buffer (ph 7.4) and acetic acid was used in a single operation during gradient elution.
The invention establishes a brand-new method for measuring the immune globulin IgG in the milk, effectively expands the types and types of the detection method of the immune globulin IgG in the milk, has high detection precision, provides more choices for the detection field, adopts a liquid chromatograph which has high popularization rate compared with the traditional detection, has good instrument operation stability and is easy to operate, and can effectively simplify the pretreatment step of detecting samples, thereby improving the detection efficiency.
The above description is only for the specific embodiments of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art can easily conceive of the changes or substitutions within the technical scope of the present invention, and all the changes or substitutions should be covered within the scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the claims.
Claims (3)
1. A method for detecting immunoglobulin igG in cow milk by high performance liquid chromatography is characterized by comprising the following steps: the high performance liquid chromatography detection method for the immunoglobulin igG in the cow milk comprises the following steps:
s1, preparing a sample, namely accurately weighing milk to be detected, adding a sodium phosphate buffer solution into the milk for dilution, then performing centrifugal separation on the diluted milk through a centrifugal machine, standing, and finally taking a supernatant after standing and filtering the supernatant through an ionic membrane to obtain a finished product for later use;
s2, detecting the chromatographic column, namely selecting a chromatographic column-Bio Protein G column, adjusting the chromatographic column-Bio Protein G column to 10-45 ℃ at a constant speed, taking 8-15 mu L of the sample prepared in the step S1, adding the sample into the chromatographic column-Bio Protein G column at a constant speed of 0.3-0.6 mL/min, adding a sodium phosphate buffer solution (pH 7.4) with the concentration of 50mmol/L and acetic acid with the concentration of 0.5mol/L into the chromatographic column-Bio Protein G column at a constant speed, and performing gradient elution operation, namely performing detection operation in the gradient elution process.
2. The method for detecting the immunoglobulin igG in cow's milk by high performance liquid chromatography according to claim 1, wherein the method comprises the following steps: in the step S1, the use ratio of the cow milk to the sodium phosphate buffer solution is 1: 2-10.
3. The method for detecting the immunoglobulin igG in cow's milk by high performance liquid chromatography according to claim 1, wherein the method comprises the following steps: in the S2, only one of sodium phosphate buffer (pH7.4) and acetic acid is used in a single operation during gradient elution.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202011085067.1ACN112461943A (en) | 2020-10-12 | 2020-10-12 | High performance liquid chromatography detection method for immunoglobulin igG in cow milk |
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| CN202011085067.1ACN112461943A (en) | 2020-10-12 | 2020-10-12 | High performance liquid chromatography detection method for immunoglobulin igG in cow milk |
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1642976A (en)* | 2002-03-25 | 2005-07-20 | 阿默森生物科学有限公司 | A mutated immunoglobulin-binding protein |
| US20180362595A1 (en)* | 2008-12-24 | 2018-12-20 | Emd Millipore Corporation | Caustic stable chromatography ligands |
| CN109633053A (en)* | 2018-12-19 | 2019-04-16 | 杭州奕安济世生物药业有限公司 | The detection method and system of cell culture protein expression quantity and the protein aggregation scale of construction |
- 2020
- 2020-10-12CNCN202011085067.1Apatent/CN112461943A/enactivePending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1642976A (en)* | 2002-03-25 | 2005-07-20 | 阿默森生物科学有限公司 | A mutated immunoglobulin-binding protein |
| US20180362595A1 (en)* | 2008-12-24 | 2018-12-20 | Emd Millipore Corporation | Caustic stable chromatography ligands |
| CN109633053A (en)* | 2018-12-19 | 2019-04-16 | 杭州奕安济世生物药业有限公司 | The detection method and system of cell culture protein expression quantity and the protein aggregation scale of construction |
Non-Patent Citations (4)
| Title |
|---|
| PATRICK T. HOLLAND等: "Determination of Soluble Immunoglobulin G in Bovine Colostrum Products by Protein G Affinity Chromatography_Turbidity Correction and Method Validation", 《JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY》* |
| S P FULTON等: "Antibody quantitation in seconds using affinity perfusion chromatography", 《BIOTECHNIQUES》* |
| ZHE YAN等: "Chromatographic behavior of mouse serum immunoglobulin G in protein G perfusion affinity chromatography", 《JOURNAL OF CHROMATOGRAPHY B》* |
| 杨祖英等: "高效亲和色谱法测定初乳素免疫球蛋白IgG", 《卫生研究》* |
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